Preservation of living biological materials is needed for a huge range of endeavors in biomedicine, spanning medical research and drug discovery, organ and tissue transplantation, cell-based therapies, fertility and regenerative medicine, emergency preparedness, and trauma care. Cell preservation has historically relied on one-size-fits-all cryopreservation approaches that often lead to significant cell death, apoptosis and batch-to- batch variation. We propose development of technology using an approach based on nature in combination with existing preservation methods using human vascular endothelial cells. Ultimately this technology can be scaled up for tissues and possibly whole organs. Our approach is to use an isochoric (constant volume) pressure system to achieve thermodynamically stable non-frozen preservation of biological systems at high sub-zero temperatures that is augmented with solution formulations based upon strategies employed by supercooling and hibernating animals in nature. The objective of this grant is to identify cryopreservation solution formulations and protocols employing isochoric pressure (<95MPa) at temperatures ranging from -5 to -20C that are compatible with cell survival under these conditions. The work is divided into 3 specific aims: In aim 1 our subaward team at University of California, Berkeley, will perform thermodynamic profiling of a series of candidate cryostasis cocktails and protocols that will limit pressure to <95MPa and allow cooling to temperatures as low as -20C without complete freezing. In aim 2 the lead conditions that ensures a potential non-frozen state will be evaluated with human vascular endothelial cells using a combinatorial, high throughput approach. Finally, in aim 3 the newly defined cryostasis cocktails and optimal cooling/warming rates from the earlier aims will be used to test cell recovery after isochoric storage as a function of temperature, pressure and storage duration. Following demonstration of feasibility using this innovative approach to cryopreservation we will plan Phase II SBIR studies in which further optimization is performed and cryopreservation of various cells, tissues and organs of research and medical interest are evaluated using our technology.